Nakazawa Yozo, Matsuda Kazuyuki, Kurata Takashi, Sueki Akane, Tanaka Miyuki, Sakashita Kazuo, Imai Chihaya, Wilson Matthew H, Koike Kenichi
Department of Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621, Japan.
Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
J Hematol Oncol. 2016 Mar 16;9:27. doi: 10.1186/s13045-016-0256-3.
Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML.
We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34(+) cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34(+) cells.
GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34(+) cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34(+) cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34(+) cell proliferation, particularly CD34(+)CD38(-) cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34(+) cell colony growth.
Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.
青少年骨髓单核细胞白血病（JMML）是一种致命的儿童早期骨髓增生异常/骨髓增殖性肿瘤。JMML患者在粒细胞-巨噬细胞集落刺激因子（GM-CSF）受体（GMR，CD116）信号通路中存在相互排斥的基因异常。异基因造血干细胞移植是目前JMML唯一的治愈性治疗选择；然而，疾病复发是治疗失败的主要原因。我们研究了使用靶向GMR的嵌合抗原受体（CAR）对JMML进行过继性免疫治疗。
我们构建了一种新型CAR，它能够通过其配体GM-CSF与GMR结合，并从三名健康供体和两名JMML患者中产生基于猪尾巴转座子的GMR CAR修饰的T细胞。我们进一步评估了GMR CAR T细胞对六名JMML患者（两名NRAS突变、三名PTPN11突变和一名7号染色体单体）的白血病CD34(+)细胞以及正常CD34(+)细胞的抗增殖潜力。
来自健康供体的GMR CAR T细胞抑制了MO7e细胞的细胞因子依赖性生长，但不抑制K562和Daudi细胞的生长。健康的GMR CAR T细胞与五名JMML患者的CD34(+)细胞以1:1和1:4的效应细胞与靶细胞比例共培养2天，无论基因异常如何，均显著降低了总集落生长。此外，来自一名未移植患者和一名移植患者的GMR CAR T细胞在发病时抑制各自JMML CD34(+)细胞的增殖，其程度与健康的GMR CAR T细胞相当。GMR CAR T细胞的7天共培养导致JMML CD34(+)细胞增殖明显受到抑制，特别是对AGM-S3细胞上干细胞因子和血小板生成素刺激的CD34(+)CD38(-)细胞增殖的抑制。同时，GMR CAR T细胞对正常CD34(+)细胞集落生长没有影响。
基于配体的GMR CAR T细胞可能对JMML中的干细胞和祖细胞具有抗增殖作用。
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